Using current technologies, embryonic atrial-, ventricular- and nodal-like cardiomyocytes can be differentiated non-specifically from hESCs6-9. Swine transplantation studies show that implanted hESC derived cardiomyocytes have pace making activities, a potential cause of ventricular arrhythmias10. The application of hESCs in myocardial repair is hampered by this cardiac subtype heterogeneity of hESC-derived cardiomyocytes1. To direct differentiation of hESCs into a desired cardiac subtype, the mechanisms of cardiac subtype specification have to be uncovered. Although several growth factors, such as activin A, bone morphogenetic protein 4 (BMP4), wnt-3a, basic fibroblast growth factor (bFGF) and dickkopf homolog 1 (DKK1), have been identified to promote cardiogenesis, and are used in several hESC cardiac differentiation protocols7,8,11, there is no evidence up to date showing that these or other growth factors regulate the cardiac subtype specification during hESC differentiation. Identifying the key regulators of cardiac subtype specification is critical for reducing the heterogeneity of hESC-derived cardiomyocyte population which will be important for its later use in regenerative medicine or as drug test systems6,12.
Bone morphogenetic protein (BMP) signaling is tightly controlled during mesoderm and heart development. In mouse embryos, the BMP antagonist Noggin is transiently but strongly expressed in the cardiac crescent at embryo day E7.5 to E8.013. Dkk1, a potent inducer of heart development8,14 15 synergizes with BMP antagonism to specify heart tissue in non-cardiogenic mesoderm from Xenopus embryos16. It has been shown that long term treatment of hESCs with BMP4 induces trophoblast-like cell differentiation17, while short term treatment initiates mesoderm formation18. Together, these results suggest that inhibiting BMP signaling after mesoderm formation facilitates cardiac development.
Retinoic acid (RA) signaling not only restricts the cardiac progenitor pool, and exposure of the anterior lateral plate mesoderm of zebrafish embryos to the RA antagonist BMS-189453 causes uncommitted lateral mesodermal cells to become myocardial progenitors19, but also regulates anterior-posterior polarization of the heart20. Chicken transplantation studies have revealed that the cardiogenic mesoderm from HH stages 4-6, originally fated to be atria, is competent to develop into functional ventricles and vice versa21,22. RA treatment of HH stage 4 cardiogenic tissue activates the expression of the atrium-specific gene AMHC1 in anterior progenitors fated to develop into out-flow track tissues23. Furthermore, in both mouse and chicken embryos, inhibition of RA signaling within critical periods produces embryos with oversized ventricles and smaller or missing atria, and exogenous addition of RA results in reverted phenotypes5,24. Furthermore, studies with mouse embryonic stem cells indicated retinoic acid promotes the expression of atrial related genes25.